Metabolic signaling and energetic environment in the nucleus is critical for cell division and initiation of tissue regeneration after injury. However energy supply routes to nuclear ATP-dependent processes and metabolic signaling circuits that govern cardiomyocyte cell cycle are unknown. Our studies demonstrate that adenylate kinase (AK)-phosphotransfer (2ADP<->ATP+AMP) plays a major role in metabolic signaling and transmission of high-energy phosphoryls from mitochondria to the nucleus to support nuclear transport. Preliminary studies using genetic and siRNA approaches indicate that the AK isoform network is critical for metabolic reprogram- ming facilitating stem cell cardiac differentiation. W have discovered that during cell cycle cytosolic AK1 trans- locates to the nucleus and associates with mitotic spindles to provide energy for cell division. However, AK1 translocation to the nucleus doesn't occur in mitotically arrested adult cardiomyocytes. Furthermore, we have discovered that deficiency of the AK2 isoform, which is localized in mitochondria, arrests stem cell develop- mental programming and is embryonically lethal. Using 18O-labeling technology we demonstrate that heart re-generative capacity depends on AK2 expression and dynamics of AMP-signaling through AK-AMP-AMPK axis which is a part of p53/p21/cyclin metabolic checkpoint regulating G1/S cell cycle transition. This highlights the significance of AK isoform and AMP-signaling network in regulating nuclear energetics and cell cycle. However molecular mechanisms of AK translocation to the nucleus and association with mitotic spindle and cytokinesis apparatus and the significance of AK and AMP-signaling in energy support of cell cycle, cardiomyocyte renewal and heart regeneration are unknown. Objective/Hypothesis: Based on new discoveries we will test hypothesis that nuclear translocation of AK isoforms and AMP-signaling is critical for the energetics of the cardiomyocyte cell cycle, and that AK-AMP-AMPK signaling axis is a key part of G1/S metabolic checkpoint licensing cardiomyocyte renewal and heart regeneration. The Specific Aims will determine: Aim #1 The significance of the AK isoforms in cardiomyocyte nuclear energetics and energy support of cell cycle machinery and AMP-signaling dependent metabolic checkpoint regulating heart regenerative potential. Aim #2 Molecular mechanisms of cell cycle dependent translocation of AK isoforms to the cell nucleus and association with mitotic spindles and cytokinesis machinery and the role in integration of mitochondrial and nuclear energetic processes. Aim #3 Mechanisms regulating AK isoform expression, cytosolic-nuclear distribution and AMP signaling by metabolic and growth factors in order to promote nuclear energetics and metabolic checkpoint facilitating stem cell cardiac differentiation and adult cardiomyocyte cell cycle required for heart regeneration. The expected outcome and the novelty of this application will be in defining for the first time molecular mechanisms governing nuclear energetics and AMP-signaling circuits during cell cycle and cell differentiation critical for cardiogenesis, heart renewal and regeneration.